Ubiquitylation, the covalent attachment of the small protein ubiquitin to other proteins, regulates a host of cellular processes. Protein ubiquitylation has become a synonym for protein degradation, and most of the current research is focused on the role of ubiquitin in targeting proteins for degradation by the 26S proteasome. However, we are beginning to appreciate that a number of proteins are regulated by ubiquitylation in a proteolysis-independent manner. Recent system-wide experiments suggest that only little more than fifty percent of ubiquitylated proteins are efficiently degraded by the proteasome, implying that protein ubiquitylation has widespread signaling functions outside the proteasome pathway. Molecular understanding of these proteolysis-independent ubiquitin signals will be important for basic biomedical research and development of therapeutics targeting the ubiquitin system. Some of the key questions are: Why are some ubiquitylated proteins degraded and others are not? How can ubiquitylation directly affect protein activity? What are the mechanisms of direct protein regulation by ubiquitylation and what are the components mediating regulation? We analyze a system regulated by the cullin-RING ubiquitin ligase complex SCFMet30, which connects metabolic stress to cell cycle regulation. This pathway is particularly suited to probe non-proteolytic signals of ubiquitylation because the same ligase modifies different substrate proteins with the same lysine-48 linked ubiquitin chain, yet some substrates are labeled for degradation while other substrates are regulated in a proteolysis-independent manner. This proposal builds on a plethora of tools available to analyze biochemistry and physiology of this pathway and will address (i) how regulated ubiquitin-binding domains can dictate signal identity and switch between ubiquitin chains signaling for degradation and non-proteolytic regulation (Aim 1); (ii) how a polyubiquitin chain can directly regulate transcription factor activity (Aim 2) and (iii) how ubiquitylation induces active disassembly/remodeling of multisubunit protein complexes to modulate their activities (Aim 3). Ubiquitylation affects many important cellular processes and has been linked to a number of human diseases including cancer, neurodegeneration, and retroviral infection. A contribution of proteolysis-independent ubiquitylation in these diseases is emerging and it will be important to understand the mechanism behind this regulation to design diagnostic tools and treatment strategies. This proposal aims to achieve detailed mechanistic insight into proteolysis-independent ubiquitin signals and to define the concepts of these regulatory ubiquitylation pathways.